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BIONEXGEN Overview

Edition 6, Jan 2014

Developing the Next Generation of Biocatalysts for Industrial Chemical Synthesis A Framework 7 supported project this 6th edition of the newsletter provides an update on project activities and highlights some of the exploitable results from the project.

BIONEXGEN – Perspectives and Outlook BIONEXGEN was funded by the EU-FP7 programme as a ‘Flagship Project’ with the overall aim of developing novel biocatalytic processes for chemical manufacture in the future. By bringing together some of the leading academic and industrial research groups from across Europe, and organizing these groups into focused work-packages, we envisaged that we could tackle some of the key underpinning problems and challenges that currently limit the more widespread application of biotechnology in the chemical industry. Projects within BIONEXGEN addressed the full range of activities required to implement a new biocatalytic process including enzyme discovery, biocatalyst engineering, high-throughput screening, enzyme immobilization, preparative scale transformations and life-cycle analysis. Within BIONEXGEN there was a degree of focus on specific platform chemicals (e.g. amines, fatty acids, carbohydrates) but these merely acted as illustrative of where new advances in technology and innovation are required in order to accelerate the uptake of industrial biocatalysis in industry.

project entitled BIOOX, which commenced on 1st October 2013, will focus on the application at scale of oxygen-dependent biocatalysts for targeted chemical synthesis. Some of the important enabling studies for BIOOX were carried out within BIONEXGEN thereby ensuring a smooth transition and translation of emerging technology into ongoing research programmes.

I believe that BIONEXGEN has been a major success, delivering on almost all aspects of the project which were initially envisaged and planned. During the lifetime of the project new products have been created (e.g. immobilized biocatalysts from the SMEs Lentikat’s a.s. and CLEA Technologies BV) and a new spin-out company has been spawned offering screening kits for biocatalysis (Discovery Biocatalysts Ltd). Patents have been filed together with a large number of publications in high-impact journals. PhD students and postdoctoral fellows have received specialist training such that they are now well placed to enter the chemical industry equipped with the requisite set of skills. Selected biocatalytic processes have been moved from the laboratory to initial scale-up in order to understand the factors which need to be further improved for commercial application

(e.g. expression, protein production, substrate tolerance of the biocatalyst). This progression to scale-up enables key data to be obtained which can be fed back into life-cycle analysis to benchmark new biocatalytic processes. Inevitably at the end of a project such as BIONEXGEN there are many unanswered questions and new challenges. For example, the work-package devoted to (chiral) amine synthesis has led to the discovery and development of two new biocatalyst platforms [(R)-selective amine oxidase and (R)-/(S)-imine reductases] which now require further research in order to improve their activity, stability etc. such that they can soon be applied in industrial biocatalysis. These new biocatalytic platforms will be taken forward within future projects through collaboration with industry. Additionally, a new EU-FP7

I would like to finish by thanking all of those who participated in BIONEXGEN, helping to make it a highly enjoyable and stimulating research programme. I am also very grateful for the guidance and advice provided by the Project Officer and the European Commission. Nicholas J Turner, Coordinator (The University of Manchester) 21 Jan 2014

Dr Rachel Heath, BIONEXGEN Researcher


BIONEXGEN Overview Introduction

Contents:

From the BIONEXGEN project manager...

01 BIONEXGEN – Perspectives and Outlook

In March 2011, one month after the start of the project, the kickoff event for BIONEXGEN was held in Brussels. The meeting was opened by Alfredo Aguilar Romanillos, Head of Unit, and Maria Fernandez Gutierrez, the Project Officer, representing Unit E2 – Biotechnologies, DG RTD of the European Commission. In opening, it was highlighted that BIONEXGEN was unique in scope, receiving the largest budget to date of any FP7 project in the KBBE area, bringing together 17 academic, SME, and large Industrial partners to develop the Next Generation of Biocatalysts for Industrial chemical Synthesis. Expectations for BIONEXGEN were therefore high, in recognition of the ambitious targets set out in the proposal and the potential to deliver the highest quality research with significant tangible benefits to the EU Industrial Biotechnology community.

02 Introduction 03 BIONEXGEN Overview 04 Exploitable Results 06 In the Press 10 Complete list of publication

In December 2013, at the Crowne Plaza Brussels Airport, BIONEXGEN hosted events showcasing the impact of EU funding on Industrial Biotechnology research in Europe. Drawing speakers, exhibitors, and delegates from academic and industrial research, IB end-users, funding bodies, and industry groups, this was an opportunity to present the benefits and outputs of FP7 projects, but also to look forward to the future of Industrial Biotechnology innovation, with the EU as a global leader in the field. The next generation of biocatalysis for industrial chemical synthesis was a technical dissemination session, featuring work from past and current projects including BIONEXGEN, KYROBIO (Grant No. 289646), AMBIOCAS (Grant No. 245144), BIOTRAINS (Grant No. 238531), CHEM21 (IMI Joint Technology Initiative), BIOINTENSE (Grant No. 312148), and P4FIFTY (Marie Curie ITN 289217). Industrial Biotechnology for Europe featured presentations and discussions on the role and impact of EU funding in developing European Industrial Biotechnology. This session drew from across the range of the European IB community, from academia, to SMEs, and industrial end-users, with invited 02

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participants from The University of Manchester, BASF SE, C-Tech Innovation Ltd, Bio-Prodict BV, Prozomix Ltd, and CLEA Technologies BV. The events brought together many representatives of the community in an excellent reflection of the strong research and development network fostered around many related and interconnected FP7 and EU projects, and the role of BIONEXGEN as flagship amongst these. As the project reaches a conclusion, it is finally possible to survey its many outputs and declare the success of BIONEXGEN. BIONEXGEN finishes at the end of January 2014, and it is very clear that the initial ambition of the project has been realised. The impact of BIONEXGEN can be judged from the body of literature highlighted here and which continues to be published under the auspices of the project, through enhanced cross-sector research collaborations which will continue beyond the lifetime of this project, and most immediately by the exploitable outputs and new products, including those discussed in this publication. Mark Corbett, Project Manager (The University of Manchester)


BIONEXGEN Overview BIONEXGEN Overview

Co-ordinated by The University of Manchester, the BIONEXGEN project consortium consists of 17 partners from 9 European countries: The University of Manchester, United Kingdom The University of Stuttgart, Germany Technical University of Denmark (DTU), Denmark The Institute of Microbiology of the Czech Academy of Sciences (IMIC), Czech Republic University of Groningen, Netherlands CLEA Technologies BV, Netherlands EntreChem SL, Spain University of Oviedo, Spain GALAB Laboratories GmbH, Germany Leibniz Institute of Plant Biochemistry, Germany Austrian Centre of Industrial Biotechnology, (ACIB) Austria Royal Institute of Technology (KTH), Stockholm, Sweden LentiKats a.s, Czech Republic Slovak University of Technology, Slovakia BASF SE, Germany University College London (UCL), United Kingdom Chemistry Innovation Ltd, United Kingdom For more information on the BIONEXGEN project visit: http://bionexgen-fp7.eu/

BIONEXGEN Research is split into 8 multi-disciplinary themes: Product Areas Industrial Amine Synthesis Amines are vital for the industrial synthesis of pharmaceuticals, bulk and speciality chemicals. Renewable Resources in Novel Polymer Chemistry Polymers are by far the largest volume of chemical products on the market with strong market pull for bio-based polymers in many industries e.g. automotive, packaging, construction, cosmetics and detergents. Applications of Enzymes to Glycoscience Enzymatic methods have great synthetic appeal for this traditionally

challenging area of chemistry, producing molecules which can be used for controlling health and disease and in food and feed. Industrial Applications of Oxidases Development of efficient and robust oxidative biocatalysts and the technology for performing selective oxidations that will be valuable for use in the pharmaceutical, fine chemical and food industries.

Underpinning Technology Fermentation Science A focus on efficient production strains and high density fermentation techniques which are critical to economic performance.

Biocatalyst Supports and Chemocatalysts Integration Application of biocatalyst immobilisation technology to utilise biocatalysts in industrial chemical synthesis. Bioprocess and Chemical Engineering Process engineering research to develop and implement new biocatalytic processes in industry. Economic, Environmental and Life Cycle Analysis Developing a simplified methodology for quick and reliable quantitative assessment.

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Exploitable Results

NOVEL POLYMER CHEMISTRY

BIOPROCESS & CHEMICAL ENGINEERING

Bio-based polymers by lipase catalysis At KTH the collaboration between biochemists and polymer chemists has created a research environment (the BioPol group) in the interdisciplinary areas of biocatalysis and polymer chemistry. Research concerns the development of strategies that promote efficient and selective enzyme catalysed synthesis of functional polymer resins and the design of efficient curing technologies where these polymer resins are transferred into tailored materials.

polymer films using thiol-ene chemistry. The BioPol group has in BIONEXGEN (and in other projects) successfully demonstrated several examples of chemo-enzymatic routes toward polymer materials applied to coatings. The BioPol group at KTH has a research platform available to meet the future challenges and development requirements in the area of bio-based polymer materials. Contact: Dr Mats Martinelle KTH – Royal Institute of Technology, School of Biotechnology, Division of Industrial Biotechnology, AlbaNova University Center , SE-106 91 Stockholm, Sweden

In BIONEXGEN the BioPol group combined activities in biocatalysis, curing technology and material characterisation aiming for functional polymers and polymer cross-linked films from bio-based monomers. Functional polyesters and poly(ester-amide)s were prepared with lipase catalysis and cured into cross-linked HO

Phone: + 46 8 5537 8384 Email: matsm@biotech.kth.se

Slovak University of Technology The Laboratories of Applied Biocatalysis at the Institute of Biotechnology and Food Science, Slovak University of Technology (SUT) offer a range of expertise across the field of biocatalysis. One of the main areas studied intensively in BIONEXGEN were applications of immobilized cells and enzymes as applied biocatalysts. Immobilization of recombinant Escherichia coli overexpressing Monoamine Oxidase (MAO) as whole cell biocatalysts, along with immobilization of cell-free enzyme extracts were studied in collaboration with partners LK (LentiKats a.s.) and UNIMAN (University of Manchester). This led to the development of MAO LentiKats biocatalysts which can be extensively recycled over the course of multiple reactions.

NH2

ethanolamine

+

O

O

O

OH

O

O O

O O

O

dimethyl succinate

methyl 10-hydroxydecanoate

O O

O

O O

dimethyl sebacate

dimethyl adipate (m=3)

(m=1)

(m=7)

Immobilized CalB (spacer) O

O HO

OH

N H

O O

amide-diol

OH

O

m

10undec en-1ol

Immobilized CalB

O O

O O

O m

O

O

O O m O O N H

O

N H

O

O O p

DP=3-4

HS

Bulk Photoinitiator UV-irradiation

O

O O

O

O

SH

O

O HS

mp

Om

O

O O

SH

Example of a chemo-enzymatic route to cross-linked polymer films developed in BIONEXGEN

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Activities during BIONEXGEN also included immobilization of recombinant Îą-L-rhamnosidase produced in Pichia pastoris. Together with IMIC (Institute of Microbiology, Academy of Sciences of the Czech Republic) and LK a number of immobilized biocatalysts in form of LentiKats were developed for applications in food (wine aroma release) and pharmaceutical manufacturing (isoquercitrin production). SUT has extensive experience in the field of immobilization protocol optimization for both isolated enzymes


and microorganisms and can offer this expertise to other academic/industrial partners. Bioreactor optimization units are available at SUT for the development of protocols for every stage of biocatalyst production, including fermentation processes and production of recombinant enzymes. The Laboratories of Applied Biocatalysis at offer the additional benefits of scale-up and large-scale verification of all technologies including downstream processing, and fermentors up to 400 l with appropriate downstream units are available for these purposes. Partnership with SUT offers the facilities for production of industrial samples in kg amounts and full pilot plant process evaluation. Contact: Ing. Martin Rebros, PhD. Institute of Biotechnology and Food Science Slovak University of Technology Radlinskeho 9 812 37 Bratislava Slovakia Phone: +421 2 59 325 480 Email: martin.rebros@stuba.sk IMPROVED BIOCATALYST & ENZYME SUPPORT Immobilization into Polyvinylalcohol Matrix, LentiKatˊs a.s.

The Applications - Decolouration of Industrial Wastewater by Laccase Immobilized into PVA matrix - Wine Aroma Release by Immobilized Rhamosidase into PVA matrix Industrial Solution Lentikats Biotechnology can be utilized for immobilization of any enzymes and cells for industrial applications in food and the pharmaceutical industry, biofuel production and waste water treatment. A key output of

the BIONEXGEN project the development and testing of industrially available enzymes laccase from Trametes versicolor and recombinant α-L-rhamnosidase from Aspergilus terreus expressed in Pichia pastoris. In comparison with other immobilization methods, Lentikats Biotechnology offers several important benefits, such as relatively simple production procedure and easy separation of the Lentikats Biocatalyst from the reaction media, high enzyme activity yields after immobilization and also high catalytic biocatalyst activity. The Lentikats Biocatalyst possesses excellent physical mechanical characteristics (elasticity, low abrasion) that provide for long-term stability and biocatalyst lifetime. Moreover, the PVA is broadly biologically undegradable and possesses no detectable toxicity. It is an inexpensive matrix for immobilization of any biologically active materials with no apparent side effects on the biochemical process. From the technical point of view due to the high concentration of microorganisms or enzymes in the Lentikats Biocatalyst and the possibilities for reuse of the Lentikats Biocatalyst, a significantly shorter retention time can be adopted, which minimizes the possibility of process contamination with a subsequent increase of the process yield. Consequently, Lentikats Biotechnology industrial applications require smaller reactors, thus leading to a reduction in investment costs and increased revenue. Lentikats Biotechnology has several advantages over the other currently available techniques and a large variety of applications. Application of this technology for existing or newly designed production processes leads to a substantial increase in process yields and a reduction in operational and investment costs. Laccases belong to the interesting group of multi copper enzymes with the ability to oxidize phenolic and non-phenolic compounds, including some environmental pollutants. Because of their efficiency, laccases are of interest for a variety of chemical industries, especially the textile industry, where they are able to catalyze dye decolorization for the treatment of manufacturing byproducts. Laccases immobilized into PVA matrix for removing/selective oxidation of dyes from

aqueous solutions (wastewater) especially for use in textile industry were developed during the BIONEXGEN project. Dye decolorization by laccase (from Trametes versicolor) immobilized into PVA matrix as Lentikats Biocatalyst providing as high as 83% removal efficiency. Rhamnosidases are biotechnologically important enzymes used for derhamnosylation of natural glycosides containing terminal α-L-rhamnose. Applications of interest in the food industry include the debittering of grapefruit and other citrus juices by hydrolysis of the rhamnoside naringin, and aroma enhancement of wines and juices by hydrolysis of terpenyl glycoside precursors. Intensification of the most important characteristic of a quality wine, its aromatic fragrance, can be achieved using immobilised Rhamnosidases developed during the BIONEXGEN project, in collaboration with SUT. Recombinant α-L-rhamnosidase from Aspergilus terreus expressed in Pichia pastoris and immobilized as Lentikats Biocatalyst was developed to provide an inexpensive and stable enzymatic solution for wine aroma enhancement.

Contact us LentiKatˊs a.s. Pod Vinicí 83 471 27 Stráž pod Ralskem Czech Republic Phone: +420 255 710 680 Fax: +420 255 710 699 Email: info@lentikats.eu Web: www.lentikats.eu

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BIONEXGEN Overview In the Press

This section highlights the technically relevant publications from within the consortium over the past year.

Redesign of a Phenylalanine Aminomutase into a Phenylalanine Ammonia Lyase Bartsch, S.; Wybenga, G. G.; Jansen, M.; Heberling, M. M.; Wu, B.; Dijkstra, B. W.; Janssen, D. B. Redesign of a Phenylalanine Aminomutase into a Phenylalanine Ammonia Lyase. ChemCatChem 2013, 5, 1797–1802. Abstract: An aminomutase, naturally catalyzing the interconversion of (S)-αphenylalanine and (R)-β-phenylalanine, was converted into an ammonia lyase catalyzing the nonoxidative deamination of phenylalanine to cinnamic acid by a rational single-point mutation. It could be shown by crystal structures and kinetic data that the flexibility of the lid that covers the active site decides whether the enzyme acts as a lyase or a mutase. An Arg92Ser mutation destabilized the closed conformation of the lid structure and converted the mutase into a lyase that exhibited up to 44-fold increased reaction rates in the enantioselective deamination of (R)-β-phenylalanine. In addition, the amination rates of cinnamic acid yielding optically pure (S)-α- and (R)-β-phenylalanine were doubled. The applicability of the mutant enzyme for kinetic resolution and asymmetric amination could be shown by biocatalysis on a preparative scale.

Asymmetric Reduction of Cyclic Imines Catalyzed by a WholeCell Biocatalyst Containing an (S)-Imine Reductase Leipold, F.; Hussain, S.; Ghislieri, D.; Turner, N. J. Asymmetric Reduction of Cyclic Imines Catalyzed by a Whole-Cell Biocatalyst Containing an (S)-Imine Reductase. ChemCatChem 2013, 5, 3505– 3508. Abstract: Biocatalytic imine reduction: A whole-cell recombinant E. coli system, producing an (S)-selective imine reductase (IRED) from Streptomyces sp. GF3546, is developed. This biocatalyst is used for the enantioselective reduction of a broad range of substrates such as dihydroisoquinolines and dihydro-β-carbolines as well as iminium ions.

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Microscale methods to rapidly evaluate bioprocess options for increasing bioconversion yields: application to the ω-transaminase synthesis of chiral amines. Halim, M.; Rios-Solis, L.; Micheletti, M.; Ward, J.; Lye, G. Microscale Methods to Rapidly Evaluate Bioprocess Options for Increasing Bioconversion Yields: Application to the ω-Transaminase Synthesis of Chiral Amines. Bioprocess Biosyst. Eng. 2013, 1–11. Abstract: This work aims to establish microscale methods to rapidly explore bioprocess options that might be used to enhance bioconversion reaction yields: either by shifting unfavourable reaction equilibria or by overcoming substrate and/or product inhibition. As a typical and industrially relevant example of the problems faced we have examined the asymmetric synthesis of (2S,3R)-2-amino-1,3,4-butanetriol from L-erythrulose using the ω-transaminase from Chromobacterium violaceum DSM30191 (CV2025 ω-TAm) and methylbenzylamine as the amino donor. The first process option involves the use of alternative amino donors. The second couples the CV2025 ω-TAm with alcohol dehydrogenase and glucose dehydrogenase for removal of the acetophenone (AP) by-product by in situ conversion to (R)-1-phenylethanol. The final approaches involve physical in-situ product removal methods. Reduced pressure conditions, attained using a 96-well vacuum manifold were used to selectively increase evaporation of the volatile AP while polymeric resins were also utilised for selective adsorption of AP from the bioconversion medium. For the particular reaction studied here the most promising bioprocess options were use of an alternative amino donor, such as isopropylamine, which enabled a 2.8-fold increase in reaction yield, or use of a second enzyme system which achieved a 3.3-fold increase in yield.


In the Press

Synthesis of 9-Oxononanoic Acid, a Precursor for Biopolymers Otte, K. B.; Kirtz, M.; Nestl, B. M.; Hauer, B. Synthesis of 9-Oxononanoic Acid, a Precursor for Biopolymers. ChemSusChem 2013, 6, 2149–2156. Abstract: Polymers based on renewable resources have become increasingly important. The natural functionalization of fats and oils enables an easy access to interesting monomeric building blocks, which in turn transform the derivative biopolymers into high-performance materials. Unfortunately, interesting building blocks of medium-chain length are difficult to obtain by traditional chemical means. Herein, a biotechnological pathway is established that could provide an environmentally suitable and sustainable alternative. A multiple enzyme two-step one-pot process efficiently catalyzed by a coupled 9S-lipoxygenase (St-LOX1, Solanum tuberosum) and 9/13-hydroperoxide lyase (Cm-9/13HPL, Cucumis melo) cascade reaction is proposed as a potential route for the conversion of linoleic acid into 9-oxononanoic acid, which is a precursor for biopolymers. Lipoxygenase catalyzes the insertion of oxygen into linoleic acid through a radical mechanism to give 9S-hydroperoxy-octadecadienoic acid (9S-HPODE) as a cascade intermediate, which is subsequently cleaved by the action of Cm-9/13HPL. This one-pot process afforded a yield of 73 % combined with high selectivity. The best reaction performance was achieved when lipoxygenase and hydroperoxide lyase were applied in a successive rather than a simultaneous manner. Green leaf volatiles, which are desired flavor and fragrance products, are formed as by-products in this reaction cascade. Furthermore, we have investigated the enantioselectivity of 9/13HPLs, which exhibited a strong preference for 9S-HPODE over 9R-HPODE.

Regulation of Pichia pastoris promoters and its consequences for protein production. Vogl, T.; Glieder, A. Regulation of Pichia Pastoris Promoters and Its Consequences for Protein Production. N. Biotechnol. 2013, 30, 385–404. Abstract: The methylotrophic yeast Pichia pastoris is a widely used host for heterologous protein production. Along with favorable properties such as growth to high cell density and high capacities for protein secretion, P. pastoris provides a strong, methanol inducible promoter of the alcohol oxidase 1 (AOX1) gene. The regulation of this promoter has been extensively studied in recent years by characterizing cis-acting sequence elements and trans-acting factors, revealing insights into underlying molecular mechanisms. However, new alternative promoters have also been identified and characterized by means of their transcriptional regulation and feasibility for protein production using P. pastoris. Besides the often applied GAP promoter, these include a variety of constitutive promoters from housekeeping genes (e.g. TEF1, PGK1, TPI1) and inducible promoters from particular biochemical pathways (e.g. PHO89, THI11, AOD). In addition to these promoter sequence/function based studies, transcriptional regulation has also been investigated by characterizing transcription factors (TFs) and their modes of controlling bioprocess relevant traits. TFs involved in such diverse cellular processes such as the unfolded protein response (UPR) (Hac1p), iron uptake (Fep1p) and oxidative stress response (Yap1p) have been studied. Understanding of these natural transcriptional regulatory networks is a helpful basis for synthetic biology and metabolic engineering approaches that enable the design of tailor-made production strains.

Structure and activity of NADPHdependent reductase Q1EQE0 from Streptomyces kanamyceticus, which catalyses the R-selective reduction of an imine substrate. Rodríguez-Mata, M.; Frank, A.; Wells, E.; Leipold, F.; Turner, N. J.; Hart, S.; Turkenburg, J. P.; Grogan, G. Structure and Activity of NADPHDependent Reductase Q1EQE0 from Streptomyces Kanamyceticus, Which Catalyses the R-Selective Reduction of an Imine Substrate. ChemBioChem 2013, 14, 1372–1379. Abstract: NADPH-dependent oxidoreductase Q1EQE0 from Streptomyces kanamyceticus catalyzes the asymmetric reduction of the prochiral monocyclic imine 2-methyl-1-pyrroline to the chiral amine (R)-2-methylpyrrolidine with >99% ee, and is thus of interest as a potential biocatalyst for the production of optically active amines. The structures of Q1EQE0 in native form, and in complex with the nicotinamide cofactor NADPH have been solved and refined to a resolution of 2.7 Å. Q1EQE0 functions as a dimer in which the monomer consists of an N-terminal Rossmanfold motif attached to a helical C-terminal domain through a helix of 28 amino acids. The dimer is formed through reciprocal domain sharing in which the C-terminal domains are swapped, with a substrate-binding cleft formed between the N-terminal subunit of monomer A and the C-terminal subunit of monomer B. The structure is related to those of known β-hydroxyacid dehydrogenases, except that the essential lysine, which serves as an acid/base in the (de) protonation of the nascent alcohol in those enzymes, is replaced by an aspartate residue, Asp187 in Q1EQE0. Mutation of Asp187 to either asparagine or alanine resulted in an inactive enzyme.

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In the Press

Priming ammonia lyases and aminomutases for industrial and therapeutic applications Heberling, M. M.; Wu, B.; Bartsch, S.; Janssen, D. B. Priming Ammonia Lyases and Aminomutases for Industrial and Therapeutic Applications. Curr. Opin. Chem. Biol. 2013, 17, 250–260. Abstract: Ammonia lyases (AL) and aminomutases (AM) are emerging in green synthetic routes to chiral amines and an AL is being explored as an enzyme therapeutic for treating phenylketonuria and cancer. Although the restricted substrate range of the wild-type enzymes limits their widespread application, the non-reliance on external cofactors and direct functionalization of an olefinic bond make ammonia lyases attractive biocatalysts for use in the synthesis of natural and non-natural amino acids, including β-amino acids. The approach of combining structure-guided enzyme engineering with efficient mutant library screening has extended the synthetic scope of these enzymes in recent years and has resolved important mechanistic issues for AMs and ALs, including those containing the MIO (4-methylideneimidazole-5-one) internal cofactor.

Substrate promiscuity of cytochrome P450 RhF O’Reilly, E.; Corbett, M.; Hussain, S.; Kelly, P. P.; Richardson, D.; Flitsch, S. L.; Turner, N. J. Substrate Promiscuity of Cytochrome P450 RhF. Catal. Sci. Technol. 2013, 3, 1490–1492.

Synthesis of ω-hydroxy dodecanoic acid based on an engineered CYP153A fusion construct Honda Malca, S.; Scheps, D.; Kuhnel, L.; Venegas-Venegas, E.; Seifert, A.; Nestl, B. M.; Hauer, B. Bacterial CYP153A Monooxygenases for the Synthesis of OmegaHydroxylated Fatty Acids. Chem. Commun. 2012, 48, 5115–5117. Abstract: A bacterial P450 monooxygenase-based whole cell biocatalyst using Escherichia coli has been applied in the production of ω-hydroxy dodecanoic acid from dodecanoic acid (C12-FA) or the corresponding methyl ester. We have constructed and purified a chimeric protein where the fusion of the monooxygenase CYP153A from Marinobacter aquaeloei to the reductase domain of P450 BM3 from Bacillus megaterium ensures optimal protein expression and efficient electron coupling. The chimera was demonstrated to be functional and three times more efficient than other sets of redox components evaluated. The established fusion protein (CYP153AM. aq.CPR) was used for the hydroxylation of C12-FA in in vivo studies. These experiments yielded 1.2 g l–1 ω-hydroxy dodecanoic from 10 g l–1 C12-FA with high regioselectivity (> 95%) for the terminal position. As a second strategy, we utilized C12-FA methyl ester as substrate in a two-phase system (5:1 aqueous/organic phase) configuration to overcome low substrate solubility and product toxicity by continuous extraction. The biocatalytic system was further improved with the coexpression of an additional outer membrane transport system (AlkL) to increase the substrate transfer into the cell, resulting in the production of 4 g l–1 ω-hydroxy dodecanoic acid. We further summarized the most important aspects of the whole-cell process and thereupon discuss the limits of the applied oxygenation reactions referring to hydrogen peroxide, acetate and P450 concentrations that impact the efficiency of the production host negatively

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Abstract: Cytochrome P450 RhF displays a high degree of substrate promiscuity, mediating a range of O-dealkylations, aromatic hydroxylations, epoxidations and asymmetric sulfoxidations. The selfsufficient nature of this CYP coupled with its ability to catalyse the oxidation of a wide range of functional groups highlights this enzyme as an excellent starting template for directed evolution and promising alternate to P450 BM3.


In the Press

Synthesis of ω-hydroxy dodecanoic acid based on an engineered CYP153A fusion construct. Scheps, D.; Honda Malca, S.; Richter, S. M.; Marisch, K.; Nestl, B. M.; Hauer, B. Synthesis of ω-Hydroxy Dodecanoic Acid Based on an Engineered CYP153A Fusion Construct. Microb. Biotechnol. 2013, 6, 694–707. Abstract: A bacterial P450 monooxygenase-based whole cell biocatalyst using Escherichia coli has been applied in the production of ω-hydroxy dodecanoic acid from dodecanoic acid (C12-FA) or the corresponding methyl ester. We have constructed and purified a chimeric protein where the fusion of the monooxygenase CYP153A from Marinobacter aquaeloei to the reductase domain of P450 BM3 from Bacillus megaterium ensures optimal protein expression and efficient electron coupling. The chimera was demonstrated to be functional and three times more efficient than other sets of redox components evaluated. The established fusion protein (CYP153AM. aq. -CPR) was used for the hydroxylation of C12-FA in in vivo studies. These experiments yielded 1.2 g l-1 ω-hydroxy dodecanoic from 10 g l-1 C12-FA with high regioselectivity (> 95%) for the terminal position. As a second strategy, we utilized C12-FA methyl ester as substrate in a two-phase system (5:1 aqueous/organic phase) configuration to overcome low substrate solubility and product toxicity by continuous extraction. The biocatalytic system was further improved with the coexpression of an additional outer membrane transport system (AlkL) to increase the substrate transfer into the cell, resulting in the production of 4 g l-1 ω-hydroxy dodecanoic acid. We further summarized the most important aspects of the whole-cell process and thereupon discuss the limits of the applied oxygenation reactions referring to hydrogen peroxide, acetate and P450 concentrations that impact the efficiency of the production host negatively.

Alkylating enzymes Wessjohann, L. A.; Keim, J.; Weigel, B.; Dippe, M. Alkylating Enzymes. Curr. Opin. Chem. Biol. 2013, 17, 229–235. Abstract: Chemospecific and regiospecific modifications of natural products by methyl, prenyl, or C-glycosyl moieties are a challenging and cumbersome task in organic synthesis. Because of the availability of an increasing number of stable and selective transferases and cofactor regeneration processes, enzyme-assisted strategies turn out to be promising alternatives to classical synthesis. Two categories of alkylating enzymes become increasingly relevant for applications: firstly prenyltransferases and terpene synthases (including terpene cyclases), which are used in the production of terpenoids such as artemisinin, or meroterpenoids like alkylated phenolics and indoles, and secondly methyltransferases, which modify flavonoids and alkaloids to yield products with a specific methylation pattern such as 7-O-methylaromadendrin and scopolamine.

The BIONEXGEN newsletter edition 5

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Complete list of publications resulting from research undertaken within the BIONEXGEN project is listed below: (1) Bartsch, S.; Wybenga, G. G.; Jansen, M.; Heberling, M. M.; Wu, B.; Dijkstra, B. W.; Janssen, D. B. Redesign of a Phenylalanine Aminomutase into a Phenylalanine Ammonia Lyase. ChemCatChem 2013, 5, 1797–1802. (2) Crismaru, C. G.; Wybenga, G. G.; Szymanski, W.; Wijma, H. J.; Wu, B.; Bartsch, S.; de Wildeman, S.; Poelarends, G. J.; Feringa, B. L.; Dijkstra, B. W.; et al. Biochemical Properties and Crystal Structure of a β-Phenylalanine Aminotransferase from Variovorax Paradoxus. Appl. Environ. Microbiol. 2013, 79, 185–195. (3) Díaz-Rodríguez, A.; Borzęcka, W.; Lavandera, I.; Gotor, V. Stereodivergent Preparation of Valuable γ- or δ-Hydroxy Esters and Lactones through One-Pot Cascade or Tandem Chemoenzymatic Protocols. ACS Catal. 0, 386–393. (4) Díaz-Rodríguez, A.; IglesiasFernández, J.; Rovira, C.; GotorFernández, V. Enantioselective Preparation of δ-Valerolactones with Horse Liver Alcohol Dehydrogenase. ChemCatChem 2013, n/a–n/a. (5) Díaz-Rodríguez, A.; Lavandera, I.; Kanbak-Aksu, S.; Sheldon, R. A.; Gotor, V.; Gotor-Fernández, V. From Diols to Lactones Under Aerobic Conditions Using a Laccase/TEMPO Catalytic System in Aqueous Medium. Adv. Synth. Catal. 2012, 354, 3405–3408. (6) Gerstorferová, D.; Fliedrová, B.; Halada, P.; Marhol, P.; Křen, V.; Weignerová, L. Recombinant α-lRhamnosidase from Aspergillus terreus in Selective Trimming of Rutin. Process Biochem. 2012, 47, 828–835. (7) Gerstorferová, D.; Fliedrová, B.; Halada, P.; Marhol, P.; Křen, V.; Weignerová, L. Recombinant α-lRhamnosidase from Aspergillus Terreus

in Selective Trimming of Rutin. Process Biochem. 2012, 47, 828–835. (8) Gudiminchi, R. K.; Geier, M.; Glieder, A.; Camattari, A. Screening for Cytochrome P450 Expression in Pichia pastoris Whole Cells by P450-Carbon Monoxide Complex Determination. Biotechnol. J. 2013, 8, 146–152. (9) Halim, M.; Rios-Solis, L.; Micheletti, M.; Ward, J.; Lye, G. Microscale Methods to Rapidly Evaluate Bioprocess Options for Increasing Bioconversion Yields: Application to the ω-Transaminase Synthesis of Chiral Amines. Bioprocess Biosyst. Eng. 2013, 1–11. (10) Heberling, M. M.; Wu, B.; Bartsch, S.; Janssen, D. B. Priming Ammonia Lyases and Aminomutases for Industrial and Therapeutic Applications. Curr. Opin. Chem. Biol. 2013, 17, 250–260. (11) Honda Malca, S.; Scheps, D.; Kuhnel, L.; Venegas-Venegas, E.; Seifert, A.; Nestl, B. M.; Hauer, B. Bacterial CYP153A Monooxygenases for the Synthesis of Omega-Hydroxylated Fatty Acids. Chem. Commun. 2012, 48, 5115–5117. (12) Leipold, F.; Hussain, S.; Ghislieri, D.; Turner, N. J. Asymmetric Reduction of Cyclic Imines Catalyzed by a WholeCell Biocatalyst Containing an (S)-Imine Reductase. ChemCatChem 2013, 5, 3505–3508. (13) O’Reilly, E.; Corbett, M.; Hussain, S.; Kelly, P. P.; Richardson, D.; Flitsch, S. L.; Turner, N. J. Substrate Promiscuity of Cytochrome P450 RhF. Catal. Sci. Technol. 2013, 3, 1490–1492. (14) Otte, K. B.; Kirtz, M.; Nestl, B. M.; Hauer, B. Synthesis of 9-Oxononanoic Acid, a Precursor for Biopolymers. ChemSusChem 2013, 6, 2149–2156. (15) Otte, K. B.; Kittelberger, J.; Kirtz, M.; Nestl, B. M.; Hauer, B. Whole-Cell One-Pot Biosynthesis of Azelaic Acid. ChemCatChem 2013, n/a–n/a. (16) Rebroš, M.; Pilniková, A.; ŠImčíková, D.; Weignerová, L.; Stloukal, R.; Křen,

V.; Rosenberg, M. Recombinant α-LRhamnosidase of Aspergillus terreus Immobilization in Polyvinylalcohol Hydrogel and Its Application in Rutin Derhamnosylation. Biocatal. Biotransformation 2013, 31, 329–334. (17) Rodríguez-Mata, M.; Frank, A.; Wells, E.; Leipold, F.; Turner, N. J.; Hart, S.; Turkenburg, J. P.; Grogan, G. Structure and Activity of NADPH-Dependent Reductase Q1EQE0 from Streptomyces kanamyceticus, Which Catalyses the R-Selective Reduction of an Imine Substrate. ChemBioChem 2013, 14, 1372–1379. (18) Šardzík, R.; Green, A. P.; Laurent, N.; Both, P.; Fontana, C.; Voglmeir, J.; Weissenborn, M. J.; Haddoub, R.; Grassi, P.; Haslam, S. M.; et al. Chemoenzymatic Synthesis of O-Mannosylpeptides in Solution and on Solid Phase. J. Am. Chem. Soc. 2012, 134, 4521–4524. (19) Scheps, D.; Honda Malca, S.; Richter, S. M.; Marisch, K.; Nestl, B. M.; Hauer, B. Synthesis of ω-Hydroxy Dodecanoic Acid Based on an Engineered CYP153A Fusion Construct. Microb. Biotechnol. 2013, 6, 694–707. (20) Sheldon, R. A. Laccase CLEAs in Textile Waste Water Treatment http://www. wtin.com/article/?articleID=2642270372 (accessed May 10, 2013). (21) Vogl, T.; Glieder, A. Regulation of Pichia Pastoris Promoters and Its Consequences for Protein Production. N. Biotechnol. 2013, 30, 385–404. (22) Wessjohann, L. A.; Keim, J.; Weigel, B.; Dippe, M. Alkylating Enzymes. Curr. Opin. Chem. Biol. 2013, 17, 229–235. (23) Wessjohann, L.; Vogt, T.; Kufka, J.; Klein, R. Prenyl- Und Methyltransferasen in Natur Und Synthese. BIOspektrum 2012, 18, 22–25. (24) Zajkoska, P.; Rebroš, M.; Rosenberg, M. Biocatalysis with Immobilized Escherichia coli. Appl. Microbiol. Biotechnol. 2013, 97, 1441–1455.

The research leading to the results described in this newsletter has received funding from the European Union Seventh Framework Programme ([FP7/2007-2013] [FP7/20072011]) under grant agreement n° 266025. The BIONEXGEN newsletter edition 5

Bionexgen's 6th Newsletter